The effect of molecular crowding with nucleotide length and cosolute structure on DNA duplex stability

The thermodynamics of DNA duplex structures in the presence of high concentrations of cosolutes in solution were investigated to discern nucleic acid structures and functions in living cells. In the presence of ethylene glycol (EG) and poly(ethylene glycol) (PEG) (MW = 200-8000), the stability of the oligomer DNA duplexes with differing nucleotide length varied, depending on the nucleotide length as well as the size of PEG. It was also revealed that the decrease of water activity is the primary factor for destabilization of the short (8-mer) duplex by addition of high molecular weight PEGs as well as low molecular weight PEGs and other low molecular weight cosolutes. In addition, the number of water molecules taken up per base pair formation was the same for all the PEGs and for 1,2-dimethoxyethane, which was greater than in the cases of glycerol, EG, 1,3-propanediol, and 2-methoxyethanol, suggesting that the solvation of nucleotides may differ, depending on the cosolute structure. These findings are useful not only for understanding nucleic acid structures and functions in cells but also for the design of oligonucleotides applicable for cells, such as antisense nucleic acids, RNAi, and DNA chips.     

Effect of solute hydrophobicity on phase behaviour in solutions of thermoseparating polymers

 Two-phase systems consisting of a polymer rich phase and polymer depleted phase, where the polymer is either ethyl(hydroxy ethyl)cellulose (EHEC) or Ucon (a random copolymer of ethylene oxide and propylene oxide), have been studied. Both of these polymers can be separated from an aqueous solution by either temperature increase or addition of cosolutes. The polymers are thermoseparating and phase separate in water solutions at the cloud point temperature. Two types of EHEC have been studied: one with a cloud point at 60 °C and the other at 37 °C. The Ucon polymer used in this study has a cloud point at 50 °C. Ternary phase diagrams of polymer/water/cosolute systems have been investigated. When a strongly hydrophilic or hydrophobic cosolute is added to an EHEC- or Ucon–water solution, a phase separation occurs already at, or below, room temperature. As cosolutes, hydrophobic molecules like phenol, butyric and propionic acid, and hydrophilic molecules like glycine, ammonium acetate, sodium carboxylates (acetate to valerate), were studied. The polymer rich phase formed when mixing polymer, water and cosolute was strongly enriched or depleted with hydrophobic or hydrophilic cosolutes, respectively. The two phase region increased for propionic acid, butyric acid and phenol as a result of increased cosolute hydrophobicity. The opposite occurred in the series sodium acetate, sodium butyrate and sodium valerate. The effect of temperature on the phase behaviour has also been investigated. Model calculations based on Flory–Huggins theory of polymer solutions are presented, in form of a phase diagram, which semiquantitatively reproduce some experimental results. Received: 5 July 1996 Accepted: 4 November 1996     

Molecular dynamics studies of ion distributions for DNA duplexes and DNA clusters: salt effects and connection to DNA melting

We present extensive molecular dynamics simulations of the ion distributions for DNA duplexes and DNA clusters using the Amber force field with implicit water. The distribution of ions and the electrostatic energy of ions around an isolated DNA duplex and clusters of DNA duplexes in different salt (NaCl) concentrations over the range 0.2-1.0 mol/L are determined on the basis of the simulation results. Using the electrostatic energy profile, we determine a local net charge fraction phi, which is found to increase with increasing of salt concentration. For DNA clusters containing two DNA duplexes (DNA pair) or four DNA duplexes, phi increases as the distance between the duplexes decreases. Combining this result with experimental results for the dependence of the DNA melting temperature on bulk salt concentration, we conclude that for a pair of DNA duplexes the melting temperature increases by 5-10 K for interaxis separations of 25-40 A. For a cluster of four DNA duplexes, an even larger melting temperature increase should occur. We argue that this melting temperature increase in dense DNA clusters is responsible for the cooperative melting mechanism in DNA-linked nanoparticle aggregates and DNA-linked polymer aggregates.     

Characterization of thermally sensitive interactions in aqueous mixtures of hydrophobically modified hydroxyethylcellulose and cyclodextrins

Effects of beta-cyclodextrin (beta-CD) or hydroxypropyl-beta-cyclodextrin (HP-beta-CD) addition and temperature on thermodynamic, rheological, and structural features of semidilute solutions of hydroxyethylcellulose (HEC) and its hydrophobically modified analogue (HM-HEC) are reported. Differential scanning calorimetric (DSC) measurements revealed a thermally induced crystal melting transition of beta-CD at high concentrations in solutions of HEC and HM-HEC. No transition with HP-beta-CD was observed in aqueous solution. Viscosity results indicated that at a cosolute concentration of 2 mm, the beta-CD units are threaded onto hydrophobic tails of HM-HEC (C16 groups) to form columnar structures. This arrangement is more effective in the encapsulation of the hydrophobic chains than the monomer hydrophobic deactivation accomplished by the HP-beta-CD units. At cosolute concentrations above 8 mm, no further decoupling of the hydrophobic interactions occurs for any of the cosolutes. Small-angle neutron scattering (SANS) experiments on HM-HEC/beta-CD mixtures suggest that the large-scale association structures in HM-HEC/D(2)O solutions are reduced upon addition of beta-CD, and an interesting temperature effect is observed at 2 mm beta-CD addition. At high beta-CD concentrations and low temperatures, the formation of large beta-CD clusters or crystallites generates cross-links in the HEC and HM-HEC networks, resulting in a viscosity enhancement of several orders of magnitude. This strong temperature effect is not reflected in the structural features probed by SANS.     

Synthesis and RNA-selective hybridization of α-l-ribo- and β-d-lyxo-configured oligonucleotides

Three α-l-ribofuranosyl analogues of RNA nucleotides (α-l-RNA analogues) have been synthesized and incorporated into oligonucleotides using the phosphoramide approach on an automated DNA synthesizer. The 4′-C-hydroxymethyl-α-l-ribofuranosyl thymine monomer was furthermore synthesized. Relative to the unmodified duplexes, incorporation of a single α-l-RNA monomer into a DNA strand leads to reduced thermal stability of duplexes with DNA complements but unchanged thermal stability of duplexes with RNA complements, whereas incorporation of more than one α-l-RNA monomer lead to moderately decreased thermal stability also of duplexes with RNA complements. Efficient hybridization with an RNA complement and no melting transition with a DNA complement were observed with stereoregular chimeric oligonucleotides composed of a mixture of α-l-RNA and affinity enhancing α-l-LNA monomers (α-l-ribo-configured locked nucleic acid). Furthermore, duplexes formed between oligodeoxynucleotides containing an α-l-RNA monomer and complementary RNA were good substrates for Escherichia coli RNase H. RNA-selective hybridization was also achieved by the incorporation of 1-(4-C-hydroxymethyl-β-d-lyxofuranosyl)thymine monomers into a DNA strand, whereas stable duplexes were formed with both complementary DNA and RNA when these monomers were incorporated into an RNA strand.     

Ion‐Mediated Polymerase Chain Reactions Performed with an Electronically Driven Microfluidic Device

The polymerase chain reaction (PCR) is a powerful method for exponentially amplifying very low amounts of target DNA from genetic, clinical, and forensic samples. However, the heating and cooling steps in PCR largely hamper the miniaturization of thermocyclers for on‐site detection of pathogens and point‐of‐care tests. Herein, we devise an ion‐mediated PCR (IM‐PCR) strategy by exploiting ion‐induced DNA denaturation/renaturation cycles. DNA duplexes are effectively denatured in alkaline solutions; whereas, the denatured single‐stranded DNA strands readily reform duplexes at neutral pH. By using an integrated microchip that can programmably control the solution pH simply switching the potential in a range of several hundred millivolts, we can trigger IM‐PCR at a constant temperature. Analogously to thermal cycling, 30 cycles of pH‐induced denaturation/renaturation were used to amplify protein DNA fragments as confirmed by DNA sequencing. We anticipate that this portable, low‐cost, and scalable IM‐PCR holds great promise for widespread biological, clinical, and environmental applications.     

Balance of enthalpy and entropy in depletion forces

Solutes added to solutions often dramatically impact molecular processes ranging from the suspension or precipitation of colloids to biomolecular associations and protein folding. Here we revisit the origins of the effective attractive interactions that emerge between and within macromolecules immersed in solutions containing cosolutes that are preferentially excluded from the macromolecular interfaces. Until recently, these depletion forces were considered to be entropic in nature, resulting primarily from the tendency to increase the space available to the cosolute. However, recent experimental evidence indicates the existence of additional, energetically-dominated mechanisms. In this review we follow the emerging characteristics of these different mechanisms. By compiling a set of available thermodynamic data for processes ranging from protein folding to protein–protein interactions, we show that excluded cosolutes can act through two distinct mechanisms that correlate to a large extent with their molecular properties. For many polymers at low to moderate concentrations the steric interactions and molecular crowding effects dominate, and the mechanism is entropic. To contrast, for many small excluded solutes, such as naturally occurring osmolytes, the mechanism is dominated by favorable enthalpy, whereas the entropic contribution is typically unfavorable. We review the available models for these thermodynamic mechanisms, and comment on the need for new models that would be able to explain the full range of observed depletion forces.     

Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins.

The binding of a series of PNA and DNA probes to a group of unusually stable DNA hairpins of the tetraloop motif has been observed using absorbance hypochromicity (ABS), circular dichroism (CD), and a colorimetric assay for PNA/DNA duplex detection. These results indicate that both stable PNA-DNA and DNA-DNA duplexes can be formed with these target hairpins, even when the melting temperatures for the resulting duplexes are up to 50 degrees C lower than that of the hairpin target. Both hairpin/single-stranded and hairpin/hairpin interactions are considered in the scope of these studies. Secondary structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic obstacles to hybridization imposed by both target and probe secondary structure are significant concerns for the continued development of antisense agents and especially diagnostic probes.     

Pyrosequencing trade mark technology at elevated temperature

To date, the Pyrosequencing trade mark technology has been performed at 28 degrees C due to the low thermostability of the firefly luciferase. In this study, firefly luciferase was stabilized in the presence of glycine betaine, allowing DNA sequencing at 37 degrees C. By increasing the temperature to 37 degrees C, false signals due to primer-dimers and loop-structures were decreased significantly. In addition, a combination of (i) replacing the natural dGTP with 7'deaza-dGTP in the polymerase chain reaction (PCR), (ii) 1.6 M glycine betaine, and (iii) an increase of the temperature to 37 degrees C enabled us to sequence a DNA template with the initial sequence 3'-ATGGCCCGGGGGGGAGCTCCA em leader 5'. Furthermore, we describe a method to analyze if a primer forms a primer-dimer with extendable 3'-ends.     

Synthesis and Properties of Carbocyclic 5′- Nor Oligodeoxynucleotides as Potential Antisense Molecules

We have synthesized carbocyclic 5'-nor oligodeoxynucleotides with a shortened overall chain length lacking the 5'-methylene. Their hybridization properties with DNA and RNA were investigated by UV and CD melting curves. These oligomers formed unstable duplexes with DNA, but formed stable duplexes with RNA selectively. In addition, these oligomers were very stable against nucleases.     

Melting thermodynamics of oligonucleic acids conjugated with relatively solvophobic linear polymers: A coarse‐grained molecular simulation study

Oligonucleic acids (ONAs), such as DNA and RNA, are used in various biotechnology and nanotechnology applications due to their ability to form a double helix that is stable at low temperature and melts at high temperatures. The melting temperature (T_m) of ONA duplexes can be tuned by the ONA composition, sequence, length and concentration, solvent quality, and salt concentration and by conjugation to other macromolecules. In this article, we use coarse‐grained (CG) molecular simulations to study ONAs conjugated with linear homopolymers that are relatively more solvophobic than the ONA. We study charged and stiff 8‐mer ONAs (e.g., DNA) and neutral and flexible 8‐mer ONAs (e.g., peptide nucleic acids or PNA), and vary the composition (or G‐C content) and sequence of ONA, conjugated homopolymer lengths and solvent quality for the polymer. For neutral and flexible ONAs, as the solvent quality worsens for the polymer, the ONA melting temperature increases from that of unconjugated ONA. The melting curves broaden with polymer length and worsening solvent quality, especially for ONAs with higher G‐C content. For charged and stiff ONAs, as the solvent quality worsens, the ONA melting temperature decreases compared to unconjugated ONA while the width of the melting curve remains the same. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2019 , 57, 1196–1208     

INTERACTION OF POLYVINYLPYRROLIDONE WITH METAL CHLORIDE AQUEOUS SOLUTIONS

Interactions of polyvinylpyrrolidone (PVP) with metal chlorides (MgCl2, CaCl2, KC1 and BaC12) have been investigated by viscometric and spectrophotometric techniques in aqueous solutions. Intrinsic viscosity [η] of (PVP) has shown a discontinuity with varying concentration of metal chlorides. The decreasing order of effectiveness of cation is K1 ＞Ca2 ＞ Mg2 ＞ Ba2 for poly(vinylpyrrolidone) solution. Changes in the absorption spectra of the cosolutes were observed in the presence of PVP in the lower limit of the UV-visible region i.e. 200-210 nm. These changes were attributed to interaction of PVP molecules with the cosolute molecules. As the concentration of the cosolute increased, a red shift in the peaks was observed, indicating an increase in interaction between PVP and cosolutes.     

The stability of intramolecular DNA quadruplexes with extended loops forming inter- and intra-loop duplexes

We have examined the formation of intramolecular quadruplex DNA structures in which the loops have been extended so as to generate short DNA duplexes. Fluorescence melting and DNase I cleavage studies show that duplexes can be formed within each loop, but that duplexes between the loops are not stable.     

Synthesis and characterization of pi-stacked phenothiazine-labeled oligodeoxynucleotides

[reaction: see text] A facile procedure for the incorporation of N-methyl phenothiazine as the terminal nucleoside in oligodeoxynucleotides is reported. The phenothiazine nucleoside analogue is synthesized and then incorporated into DNA using an automated DNA solid-phase synthesizer. Phenothiazine-labeled oligodeoxynucleotides form stable B-form duplexes with higher melting temperatures compared to unlabeled DNA duplexes.     

Characterization of a Family B DNA Polymerase from the Hyperthermophilic Crenarchaeon Ignicoccus hospitalis KIN4/I and Its Application to PCR

A family B DNA polymerase gene from the hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I was highly expressed under the control of T7lac promoter of pET-28ARG in Escherichia coli BL21-CodonPlus(DE3)-RIL cells. The produced I. hospitalis (Iho) DNA polymerase was purified by heat treatment followed by HisTrap? HP column and HiTrap? SP column chromatographies. The molecular mass of the purified Iho DNA polymerase was 88 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH for Iho DNA polymerase activity was 7.0 and the optimal temperature was 70 °C. Iho DNA polymerase was strongly activated by the presence of magnesium ion at an optimum concentration of 3 mM. The optimal concentration of KCl for Iho DNA polymerase activity was 60 mM. The half-life of the enzyme at 94 °C was about 2 h. The optimal conditions for polymerase chain reaction (PCR) were determined. Iho DNA polymerase possesses 3′?→?5′ exonuclease activity, and the fidelity of the Iho DNA polymerase was similar to that of Pfu and Vent DNA polymerases. However, Iho DNA polymerase provided more enhanced efficiency of PCR amplification than Pfu and Vent DNA polymerases. Iho DNA polymerase could successfully amplify a 2-kb λ DNA target with a 10-s extension time and could amplify a DNA fragment up to 8 kb λ DNA.     

Cosolutes effects on aqueous two-phase systems equilibrium formation studied by physical approaches

The effect of urea and sodium salts of monovalent halides on the aqueous polyethyleneglycol solution and binodal diagrams of polyethyleneglycol–potassium phosphate (polyethyleneglycol of molecular mass 1500, 4000, 6000 and 8000) were studied using different physical approaches. The effect of these solutes on the binodal diagram for polyethyleneglycol–potassium phosphate was also investigated. The cosolutes affected in a significant manner the water structured around the ethylene chain of polyethyleneglycol inducing a lost of this. The equilibrium curves for the aqueous two-phase systems were fitting very well by a sigmoidal function with two parameters, which are closely related with the cosolute structure making or breaking capacity on the water ordered.     

Kinetic evidence for hydrophobically stabilized encounter complexes formed by hydrophobic esters in aqueous solutions containing monohydric alcohols.

The pH-independent hydrolysis of four esters, p-methoxyphenyl 2,2-dichloroethanoate (1a), p-methoxyphenyl 2,2-dichloropropanoate (1b), p-methoxyphenyl 2,2-dichlorobutanoate (1c), and p-methoxyphenyl 2,2-dichloropentanoate (1d), in dilute aqueous solution has been studied as a function of the molality of added cosolutes ethanol, 1-propanol, and 1-butanol. The rate constants for the neutral hydrolysis decrease with increasing cosolute concentration. These kinetic medium effects respond to both the hydrophobicity of the ester and of the monohydric alcohol. The observed rate effects were analyzed using both a thermodynamic and a kinetic model. The kinetic model suggests a molecular picture of a hydrophobically stabilized encounter complex, with equilibrium constants K(ec) often smaller than unity, in which the cosolute blocks the reaction center of the hydrolytic ester for attack by water. The formation of these encounter complexes leads to a dominant initial-state stabilization as follows from the thermodynamic model. Decreases in both apparent enthalpies and entropies of activation for these hydrolysis reactions correspond to unfavorable enthalpies and favorable entropies of complexation, which confirms that the encounter complexes are stabilized by hydrophobic interactions.     

Synthesis and evaluation of a new non-fluorescent quencher in fluorogenic oligonucleotide probes for real-time PCR

A non-fluorescent quencher, based on the diaminoanthraquinone Disperse Blue 3, has been incorporated into oligonucleotides at the 5'-end, the 3'-end and internally as a thymidine derivative. Fluorimetry and fluorogenic real-time PCR experiments demonstrate that the quencher is effective with a wide range of fluorescent dyes. The anthraquinone moiety increases the melting temperature of DNA duplexes, thus allowing shorter, more discriminatory probes to be used. The quencher has been used in Scorpion primers and TaqMan probes for human DNA sequence recognition and mutation detection.     

On the stability of double stranded nucleic acids

We present the first pressure-versus-temperature phase diagram for the helix-to-coil transition of double stranded nucleic acids. The thermodynamic stability of a nucleic acid duplex is a complex function of temperature and pressure and strongly depends on the denaturation temperature, T(M), of the duplex at atmospheric pressure. Depending upon T(M), pressure, and temperature, the phase diagram shows that pressure may stabilize, destabilize, or have no effect on the conformational state of DNA. To verify the phase diagram, we have conducted high-pressure UV melting experiments on poly(dIdC)poly(dIdC), a DNA duplex, poly(rA)poly(rU), an RNA duplex, and poly(dA)poly(rU), a DNA/RNA hybrid duplex. The T(M) values of these duplexes have been modulated by altering the solution ionic strength. Significantly, at low salt, these three duplexes have helix-to-coil transition temperatures of 50 degrees C or less. In agreement with the derived phase diagram, we found that the polymeric duplexes were destabilized by pressure if the T(M) is < approximately 50 degrees C. However, these duplexes were stabilized by pressure if the T(M) is > approximately 50 degrees C. The DNA/RNA hybrid duplex, poly(dA)poly(rU), with a T(M) of 31 degrees C in 20 mM NaCl undergoes a pressure-induced helix-to-coil transition at room temperature. This is the first report of pressure-induced denaturation of a nucleic acid duplex and provides new insights into the molecular forces stabilizing these structures.     

Multiplex PCR with multichannel microchip electrophoresis: an ultrafast analysis for genetic diseases

A Y chromosomal polymorphic markers screening strategy using a multiplex polymerase chain reaction (PCR) and DNA microchip electrophoresis technology has recently been developed. It is a part of the human Y chromosome haplotyping system for studying Japanese population genetics and its relationship with male spermatogenic failure. This strategy is based on optimizing and modifying the primer set concentrations while keeping all other components of the PCR mixtures and conditions similar to those of a singleplex PCR. Well-balanced PCR products are obtained without changing even the DNA oligomer melting temperatures. Here, a panel of primer sets are used to amplify two groups of Y chromosome markers. The first consists of five markers and the second consists of seven markers. Both are possibly deleted in infertile men. The microchip electrophoresis technology is fast and sensitive, enables direct molecular typing of several Y chromosomal markers, and is separated by a difference of as many as six base pairs.